Template:Kyoto/Notebook/Sep 1

From 2013.igem.org

(Difference between revisions)
(→Sep 1)
 
(37 intermediate revisions not shown)
Line 2: Line 2:
===Transformation===
===Transformation===
<div class="experiment">
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
!Name||Sample||Competent Cells||Total||Plate
 +
|-
 +
|8/31 tRNA-Spinach(pSB1C3)||2&micro;L||20&micro;L||22&micro;L||CP
 +
|-
 +
|8/31 pT181 antisense(pSB1C3)||2&micro;L||20&micro;L||22&micro;L||CP
 +
|-
 +
|8/31 pT181 attenuator(pSB1C3)||2&micro;L||20&micro;L||22&micro;L||CP
 +
|-
 +
|8/31 pT181 antisense(pSB1C3)||2&micro;L||20&micro;L||22&micro;L||CP
 +
|-
 +
|8/31 pT181 attenuator(2)-DT||2&micro;L||20&micro;L||22&micro;L||CP
 +
|-
 +
|8/31 pT181 antisense-DT||2&micro;L||20&micro;L||22&micro;L||CP
 +
|-
 +
|8/31 tRNA-Spniach-DT||2&micro;L||20&micro;L||22&micro;L||CP
 +
|-
 +
|8/31 RBS-lysis2-DT||2&micro;L||20&micro;L||22&micro;L||CP
 +
|-
 +
|8/31 Pconst-RBS-tetR-DT||2&micro;L||20&micro;L||22&micro;L||Amp
 +
|-
 +
|8/31 Plac-RBS-lacZα-DT||2&micro;L||20&micro;L||22&micro;L||CP
 +
|-
 +
|8/31 Ptet-RBS-lacZα-DT||2&micro;L||20&micro;L||22&micro;L||CP
 +
|-
 +
|8/31 ||2&micro;L||20&micro;L||22&micro;L||CP
 +
|}
</div>
</div>
-
===PCR===
+
 
 +
===ligation production PCR===
<div class="experiment">
<div class="experiment">
 +
<span class="author">No name</span>
 +
{| class="wikitable"
 +
!Sample||base pair
 +
|-
 +
|8/31 tRNA Spinach(pSB1C3)||459
 +
|-
 +
|8/31 pT181 antisense(2)(pSB1C3)(EcoRI+SpeI)||415
 +
|-
 +
|8/31 pT181 attenuator(2)(pSB1C3)(XbaI+PstI)||601
 +
|-
 +
|8/31 pT181 antisense(2)(pSB1C3)(XbaI+PstI)||415
 +
|-
 +
|8/31 pT181 attenuator(2)-DT||754
 +
|-
 +
|8/31 pT181 antisense(2)-DT||577
 +
|-
 +
|8/31 tRNA Spinach-DT||596
 +
|-
 +
|8/22 pSB1C3(2)(controll)||1383
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
|94&deg;C||94&deg;C||55&deg;C||68&deg;C||--
 +
|-
 +
|5min||30s||30s||30min||30cycle
 +
|}
 +
[[File:Igku Sep1 ligation production PCR.jpg]]
</div>
</div>
-
===Master Plate===
+
 
 +
===Ligation===
<div class="experiment">
<div class="experiment">
 +
<span class="author">Nakamoto</span>
 +
{| class="wikitable"
 +
!state||colspan="2"|Vector||colspan="2"|Inserter
 +
|-
 +
|experiment||DT(EcoRI+SpeI)||5.5||8/28 RBS-lysis1(EcoRI+SpeI)2.4ng/&micro;L||2.4
 +
|}
 +
1. Samples were evaporeted used evaporator into about 7 µL.<br>
 +
2. Add Ligation High 3.5&micro:L and incubate 16&deg;C 1hour.<br>
</div>
</div>
 +
 +
===Liquid Culture===
 +
<div class="experiment">
 +
<span class="author">Tatsui,Stephane,Nakamoto</span>
 +
{| class="wikitable"
 +
!Sample||medium
 +
|-
 +
|8/21 pSB1C3(BBa_J04450)-(1)||Plusgrow medium(+CP)
 +
|-
 +
|8/21 pSB1C3(BBa_J04450)-(2)||Plusgrow medium(+CP)
 +
|-
 +
|RBS-lysis3-DT||Plusgrow medium(+CP)
 +
|}
 +
 +
===M9 Medium===
===M9 Medium===
<div class="experiment">
<div class="experiment">
-
</div>
+
<span class="author">No name</span>
 +
5xM9 liquid medium
 +
{|class="wikitable"
 +
!volume||100mL
 +
|-
 +
|NaHPO4||30g
 +
|-
 +
|KH2PO||3g
 +
|-
 +
|Nacl||0.25g
 +
|-
 +
|NH4Cl||0.5g
 +
|-
 +
|3.75% agar solution||400mL
 +
|-
 +
|1M MgSO4||0.5mL
 +
|-
 +
|2M Glucose||2.8mL
 +
|-
 +
|1% Vitamin B1||0.5mL
 +
|-
 +
|1M CaCl2||0.05mL
 +
|}
 +
1. mix 5xM9 medium and 3.75%Agar solution 400mL<br>
 +
2. Add 1M MgSO4, 2M Glucose and 1 VitaminB1 0.5mL <br>
 +
3. Add CaCl2 while shaking Erlenmeyer flask.
 +
 
===Miniprep===
===Miniprep===
<div class="experiment">
<div class="experiment">
 +
{|class="wikitable"
 +
!DNA||concentration[&micro;g/mL]||260/280||260/230
 +
|-
 +
|8/21 pSB1C3(1)||309.2||1.69||1.72
 +
|-
 +
|8/21 pSB1C3(2)||201.6||1.65||1.76
 +
|}
</div>
</div>
 +
===Kanamycin applicaton===
===Kanamycin applicaton===
<div class="experiment">
<div class="experiment">
-
</div>
+
<span class="author">Hirano</span>
 +
Add 20&micro;L Kanamycin into 80&micro;L MilliQ and apply it on a 9/1 M9 plate</div>
 +
 
===Plating===
===Plating===
<div class="experiment">
<div class="experiment">
-
</div>
+
<span class="author">Hirano</span>
 +
Apply entA-colony liquid culture (100&micro;L) on M9(Km)plate and incubate 37&deg;C</div>
 +
2 fold diluted OD 600 2.107
===Liquid Culture===
===Liquid Culture===
<div class="experiment">
<div class="experiment">
 +
<span class="author">Hirano</span>
 +
{| class="wikitable"
 +
!Sample||medium
 +
|-
 +
|9/1 entA- Master Plate-(4)||SOB Medium(+Km) 　
 +
|}
</div>
</div>

Latest revision as of 17:19, 25 September 2013

Contents

Sep 1

Transformation

No name

NameSampleCompetent CellsTotalPlate
8/31 tRNA-Spinach(pSB1C3)2µL20µL22µLCP
8/31 pT181 antisense(pSB1C3)2µL20µL22µLCP
8/31 pT181 attenuator(pSB1C3)2µL20µL22µLCP
8/31 pT181 antisense(pSB1C3)2µL20µL22µLCP
8/31 pT181 attenuator(2)-DT2µL20µL22µLCP
8/31 pT181 antisense-DT2µL20µL22µLCP
8/31 tRNA-Spniach-DT2µL20µL22µLCP
8/31 RBS-lysis2-DT2µL20µL22µLCP
8/31 Pconst-RBS-tetR-DT2µL20µL22µLAmp
8/31 Plac-RBS-lacZα-DT2µL20µL22µLCP
8/31 Ptet-RBS-lacZα-DT2µL20µL22µLCP
8/31 2µL20µL22µLCP

ligation production PCR

No name

Samplebase pair
8/31 tRNA Spinach(pSB1C3)459
8/31 pT181 antisense(2)(pSB1C3)(EcoRI+SpeI)415
8/31 pT181 attenuator(2)(pSB1C3)(XbaI+PstI)601
8/31 pT181 antisense(2)(pSB1C3)(XbaI+PstI)415
8/31 pT181 attenuator(2)-DT754
8/31 pT181 antisense(2)-DT577
8/31 tRNA Spinach-DT596
8/22 pSB1C3(2)(controll)1383
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s30min30cycle

Igku Sep1 ligation production PCR.jpg

Ligation

Nakamoto

stateVectorInserter
experimentDT(EcoRI+SpeI)5.58/28 RBS-lysis1(EcoRI+SpeI)2.4ng/µL2.4

1. Samples were evaporeted used evaporator into about 7 µL.
2. Add Ligation High 3.5&micro:L and incubate 16°C 1hour.

Liquid Culture

Tatsui,Stephane,Nakamoto

Samplemedium
8/21 pSB1C3(BBa_J04450)-(1)Plusgrow medium(+CP)
8/21 pSB1C3(BBa_J04450)-(2)Plusgrow medium(+CP)
RBS-lysis3-DTPlusgrow medium(+CP)


M9 Medium

No name 5xM9 liquid medium

volume100mL
NaHPO430g
KH2PO3g
Nacl0.25g
NH4Cl0.5g
3.75% agar solution400mL
1M MgSO40.5mL
2M Glucose2.8mL
1% Vitamin B10.5mL
1M CaCl20.05mL

1. mix 5xM9 medium and 3.75%Agar solution 400mL
2. Add 1M MgSO4, 2M Glucose and 1 VitaminB1 0.5mL
3. Add CaCl2 while shaking Erlenmeyer flask.

Miniprep

DNAconcentration[µg/mL]260/280260/230
8/21 pSB1C3(1)309.21.691.72
8/21 pSB1C3(2)201.61.651.76

Kanamycin applicaton

Hirano

Add 20µL Kanamycin into 80µL MilliQ and apply it on a 9/1 M9 plate

Plating

Hirano

Apply entA-colony liquid culture (100µL) on M9(Km)plate and incubate 37°C

2 fold diluted OD 600 2.107

Liquid Culture

Hirano

Samplemedium
9/1 entA- Master Plate-(4)SOB Medium(+Km) 　
Retrieved from "http://2013.igem.org/Template:Kyoto/Notebook/Sep_1"